Methods and kits for diagnosing and treating b-cell chronic lymphocytic leukemia

ABSTRACT

The present invention relates to methods and kits for detecting several polynucleotide sequence found to be indicative of a poor prognosis of B-CLL. All the polynucleotides are transcribed from a region on human chromosome 12p21-22. Most of the polynucleotides do not encode larger polypeptides, but may encode small peptides, they may function as RNAs. Four polynucleotides encode a novel protein, which in one preferred embodiment can be used as a cytokine, preferably as an interleukin. Furthermore the invention relates to methods and compositions for treating B-CLL in particular poor prognosis B-CLL.

All patent and non-patent references cited in the present application,are hereby incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to methods and kits for detecting severalpolynucleotide sequence found to be indicative of a poor prognosis ofB-CLL. All the polynucleotides are transcribed from a region on humanchromosome 12p21-22. Most of the polynucleotides do not encode largerpolypeptides, but may encode small peptides, they may function as RNAs.Four polynucleotides encode a novel protein, which in one preferredembodiment can be used as a cytokine, preferably as an interleukin.Furthermore the invention relates to methods and compositions fortreating B-CLL in particular poor prognosis B-CLL.

BACKGROUND OF INVENTION

B-CLL is the most common form of leukaemia in Denmark, with more than250 new cases diagnosed every year. The disease results in accumulationof CD19+CD5+CD23+ lymphocytes in the blood, bone marrow and organs ofthe patients. B-CLL cells are long-lived, slowly dividing and locked inthe G₁ phase of the cell cycle. At this time it is unknown how or whyB-CLL occurs and no cure is known for B-CLL. The application of moreaggressive treatment strategies has been hampered by the inability toidentify reproducible and reliable prognostic predictors in patientswith poor outcome in this disease. In many patients the diagnosis doesnot affect morbidity or mortality. Other patients suffer from anincurable cancer that inevitably results in death, regardless oftreatment. Until recently this latter group of patients could not beidentified at the time of diagnosis. Recently, two studies establishedthe mutational status of immunoglobulin variable region of the heavychain (Ig V_(H)) genes in B-CLL as independent prognostic markers,within each clinical stage (Damle, et al. & Hamblin, et al.). Patientswithout somatic hypermutation show much shorter survival than patientswith somatic hypermutation. FISH-studies of cytogenetic aberrations inB-CLL established specific abnormalities on chromosomes 11 (ATM), 12(?), 13 (Leu-1 and -2) and 17 (p53) as independent prognostic markers,within each clinical stage (Dohner, et al.). Very recent studies havedemonstrated that independent risk prediction, using a combined analysisof Ig V_(H) gene mutational analysis and cytogenetics, can identifysubgroups of B-CLL with median survivals ranging from less than 2.5years to more than 15 years (Krober, et al., Lin, et al., & Oscier, etal.) (see FIG. 1). Since the process of characterising the Ig VH genemutational status of an individual patient is cumbersome, it isdesirable to provide easier tests based on diagnostic markers for use inthe differential diagnosis of such cancer patients.

SUMMARY OF INVENTION

It is an object of preferred embodiments of the present invention toprovide differentially expressed transcription products, which can beused as prognostic markers of disease and give information about thedifferences in etiology between different groups of B-CLL patients.These differentially expressed transcription products are geneticmarkers that can be used in an easy assay to distinguish betweensubgroups of B-CLL patients and especially identify B-CLL patients witha poor prognosis.

This method for diagnosing a subtype of B-cell chronic lymphocyticleukaemia (B-CLL) comprises the steps of determining the presence orabsence of at least one expression product such as a transcriptionalproduct which comprise a nucleotide sequence selected from the groupconsisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15,SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 in a biological sampleisolated from a individual. As evidenced by the appended examples, thepresent inventors have determined that the expression products of thisinvention are present in one subtype of B-CLL having poor prognosis andthus of great diagnostic value and independent prognostic value. Equallyimportant, an expression product comprising a nucleotide sequenceselected from the group consisting of SEQ ID No:12, SEQ ID No:13, SEQ IDNo:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 has notbeen found in any of the other tissue types tested (see e.g. FIG. 8).

The vast majority of patients which show expression of the AMB-1 gene inform of at least one of the expression products selected from the groupconsisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15,SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 show unmutated Ig V(H) geneswhich is consistent with poor prognosis B-CLL. The presence of anexpression product of the AMB-1 gene can be determined easily usingstandard laboratory procedures and equipment. Therefore the diagnosticmethod provided by the present inventors provides an easy method ofdiagnosis as compared to the determination of the mutation status of IgV(H) genes and can furthermore give additional information about theprognosis.

Accordingly, a further object of preferred embodiments of the presentinvention is a method for determining the progress of B-CLL comprisingdetermining the amount of at least one expression product which comprisea nucleotide sequence selected from the group consisting of SEQ IDNo:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ IDNo:17 and SEQ ID No:18 in a biological sample isolated from anindividual. The method may be used e.g. for determining the efficiencyof a treatment, i.e. to see whether the amount of the expression productdecreases or increases in response to a curative treatment.

The expression products of the present invention are all transcripts ofSEQ ID NO:1 and/or SEQ ID No:5, the gene of the present invention calledAMB-1 which also encodes a novel polypeptide (SEQ ID NO:3).

A further object of preferred embodiments of the present invention is toprovide a cure and/or treatment of patients with B-CLL, in particular ofpatients with poor prognosis B-CLL such as the sub-type of B-CLL whichis characterised by the presence of an expression product of the presentinvention.

The method for treating B-CLL comprises administering to an individualwith a B-CLL diagnosis a compound capable of decreasing or inhibitingthe formation of an expression product of SEQ ID NO:1 and/or SEQ IDNO:5. This expression product preferably comprises a nucleotide sequenceselected from the group consisting of SEQ ID No:12, SEQ ID No:13, SEQ IDNo:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No: 17 and SEQ ID No: 18. Thepresent inventors believe that the presence of at least one of saidexpression products is an etiological factor in B-CLL and that thedisease can be treated or cured by inhibiting the expression of at leastone of such products and/or by inhibiting the effect of such product bye.g. rendering it inactive.

A further preferred object of embodiments of the present invention is todestroy or to eliminate the transcription of at least one expressionproduct comprising at least one nucleotide sequence selected form thegroup consisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ IDNo:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 for the treatment ofcancer, such as a poor prognosis sub-type of B-CLL.

The above destruction or elimination is obtained by applyingpolynucleotides or oligonucleotides in the form of small interfering RNAmolecules (siRNA), antisense molecules or ribozymes.

In one aspect the invention relates to a gene therapy vector capable ofinhibiting or decreasing the formation of an expression product of SEQID NO:1 and/or SEQ ID NO:5, said gene therapy vector preferably encodinga specific siRNA molecule, a specific antisense molecule or a specificribozyme being capable of decreasing or inhibiting the formation of anexpression product of SEQ ID NO:1 and/or SEQ ID NO:5. This gene therapyvector can be used for treating B-CLL based on the finding that theAMB-1 gene encoded by SEQ ID No:1 and/or SEQ ID No:5 is an etiologicalfactor in B-CLL.

Both SEQ ID No 1 which is a 20,000 nucleotide long sequence and SEQ IDNo 5 which is a 80,000 nucleotide long sequence provides severaltranscriptional products in B-CLL cells in patients with poor prognosisB-CLL. Some of the transcriptional products e.g. SEQ ID No 2 and SEQ IDNo 4 consists of two exons (SEQ ID No: 15 and SEQ ID No: 16) separatedby the same intron. Both mRNA sequences encode an open reading frame(SEQ ID No: 17) encoding a 121 amino acid peptide (SEQ ID No 3).

Accordingly, yet another object of preferred embodiments of the presentinvention relates to a novel class of polypeptides. These may bedescribed as a group of isolated polypeptides or proteins comprising oressentially consisting of the amino acid sequence of SEQ ID No. 3, or afragment thereof, or a polypeptide functionally equivalent to SEQ ID No.3, or a fragment thereof, wherein said fragment or functionallyequivalent polypeptide has at least 60% sequence identity with thepolypeptide of SEQ ID No 3. The polypeptides of the present inventionmay have interleukin or cytokine activity.

In a still further aspect the invention relates to an isolatedpolynucleotide selected from the group consisting of:

-   i) a polynucleotide comprising nucleotides of SEQ ID No 5,-   ii) a polynucleotide encoding a polypeptide having the amino acid    sequence of SEQ ID No 3,-   iii) a polynucleotide, the complementary strand of which hybridises,    under stringent conditions, with a polynucleotide as defined in any    of i) and ii).-   iv) a polynucleotide which is degenerate to the polynucleotide of    iii), and-   v) the complementary strand of any such polynucleotide.

The polypeptides encoded by the polynucleotides may furthermore

-   a) have at least 60% sequence identity with the amino acid sequence    of SEQ ID No 3 and have interleukin or cytokine activity,-   b) be recognised by an antibody, or a binding fragment thereof,    which is capable of recognising an epitope, wherein said epitope is    comprised within a polypeptide having the amino acid sequence of SEQ    ID No 3; and/or-   c) be competing with a polypeptide having the amino acid sequence as    shown in SEQ ID No 3 for binding to at least one predetermined    binding partner such as a cytokine receptor.

One further therapeutic application of the present invention is a methodof vaccination against B-CLL said method comprising immunising anindividual against a translational product of SEQ ID No:1 and/or SEQ IDNo:5. By stimulating the immune system of an individual to produceantibodies against the translational product the individual can becomeimmune towards B-CLL and/or the method can be used as part of therapy.The state of the art describes various ways of immunising an individualagainst a particular protein.

Finally, the invention provides a method for determining an increased ordecreased predisposition for B-CLL comprising determining in abiological sample from an individual a germline alteration in a targetnucleic acid sequence comprising 150,000 nucleotides, said targetnucleic acid sequence comprising at least 10 nucleotides of SEQ ID No:1and/or SEQ ID No:5. This aspect is based on the finding of theimportance of the expression product of SEQ ID No:1 and/or SEQ ID No:5,and the absence of any detectable expression product of SEQ ID No:1and/or SEQ ID No:5 in healthy tissue and in patients with good prognosisB-CLL. It is highly likely that the difference is caused by a germlinealteration. A germline alteration can be targeted by gene therapymethods and by the methods provided in the present invention.

DESCRIPTION OF DRAWINGS

FIG. 1: Overall survival of B-CLL patients by genotype (all stages) Theprognostic significance of V_(H) homology and cytogenetic aberrations isindependent of clinical stage (from Kröber et al., 2002 (4)).

FIG. 2 (a-d): Survival curves for survival or time to progression basedon AMB-1 expression or IgV_(H) mutational status respectively. Patientsare newly diagnosed, untreated B-CLL patients (n=34).

FIG. 3: Northern blotting demonstrating the expression level of AMB-1 invarious tissue samples using as a probe an 875 base pair fragment ofExon 3/Seq ID No: 16. UPN 1, 4 and 7 are unmutated B-CLL patients, UPN19, 9, 10, 13 and 21 are mutated B-CLL patients. Included are RNAsamples from normal Colon, Spleen, Bone Marrow and PBL (peripheral bloodlymphocytes) and RNA from the Ramos and Granta cell lines. Equal loadingof lanes was confirmed by re-probing with an actin probe (results notshown).

FIG. 4. Alignment of AMB1 with IL4 based on structural similarity. IL4is called d1iara in the alignment. The additional lines indicate thestructural similarity.

FIG. 5. A 3D search, where the peptide sequence has been searched forsimilarity to known protein or peptide 3D-structures.

FIG. 6. Predicted 3-D structure of AMB-1 compared to the known 3-Dstructure of human IL4. Prediction is performed using SEQ ID No:3 andthe method described in: Enhanced Genome Annotation using StructuralProfiles in the Program 3D-PSSM. Kelley L A, MacCallum R M & Sternberg MJ E (2000). J. Mol. Biol. 299(2), 499-520.

FIG. 7. Alignment of the AMB1 peptide sequence with the sequences ofIL4, IL3, IL13 and GM-CSF, based on their structures.

FIG. 8. A table showing the tissue types on the MTE array used for dotblotting of AMB-1 to check for expression in other tissue types.

FIG. 9. Schematic representation of the transcriptional products of thepresent invention compared to the genomic AMB1 sequence (1 & 5). 2 ismRNA short form (SEQ ID No 2). 4 is mRNA long form (SEQ ID No 4). 6 (SEQID No:6), 7 (SEQ ID No:7), 8 (SEQ ID No:8), 9 (SEQ ID No:9), 10 (SEQ IDNo:10) and 11 (SEQ ID No:11) are alternative transcription products allcomprising at least one nucleotide sequence selected form the groupconsisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No: 15,SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18.

FIG. 10. RT-PCR demonstrating the expression of AMB-1 in B-CLL patientsby RT-PCR. UPN1-UPN8 are unmutated patients, UPN9-UPN16 are mutatedpatients.

DETAILED DESCRIPTION OF THE INVENTION

The present invention in particular relates to polynucleotide sequencesfound to be indicative of a poor prognosis of B-CLL and new methods andcompositions for treating B-CLL in particular poor prognosis of B-CLL.An overview of the sequences disclosed by the present invention ispresent here:

SEQ ID No:1 includes 20.000 bp human genome sequence, derived from BACclone acc. no. AC063949. It includes the region encoding the mRNAscorresponding to the cDNAs described in Seq ID Nos:2, 4, 6, 7, 8, 9, 10,and 11 and possible up- and down-stream regulatory sequences. Seq ID No:1 includes a subset of the sequence described in Seq ID No:5. Thesequence is derived from human 12q21-22.

SEQ ID No:2 includes a cDNA corresponding to a putative mRNA transcriptthat includes the region encoding the peptide sequence in Seq ID No:3and up- and downstream regions. It is transcribed from the +strand ofSeq ID No:1 and Seq ID No:5 from human 12q21-22. From position 2317,this sequence is identical to Seq ID No:4.

Seq ID No:3 contains a peptide sequence encoded by some mRNAstranscribed from the region on human chromosome 12q21-22 included in SeqID No:1 and Seq ID No:5. It is encoded by the mRNA sequences identifiedas cDNAs in Seq ID Nos 02, 04, 09 and 11.

Seq ID No:4 includes a cDNA corresponding to a putative mRNA transcriptthat includes the region encoding the peptide sequence in Seq ID No:3and up- and downstream regions. It is transcribed from the +strand ofSeq ID No:1 and Seq ID No:5 from human 12q21-22.

Seq ID No:5 includes 80.000 bp human genome sequence, derived from BACclone acc. no. AC063949. It is an expansion of the genomic sequenceincluded in Seq ID No: 1. which is contained within this sequence. Itincludes the region encoding the mRNAs corresponding to the cDNAsdescribed in Seq ID Nos 02, 04, 06, 07, 08, 09, 10 and 11 and possibleup- and down-stream regulatory sequences. The sequence is derived fromhuman 12q21-22.

Seq ID No:6 corresponds to a cDNA detected by cDNA cloning,corresponding to an mRNA transcript. It includes two exons. It istranscribed from the +strand of Seq ID No:1 and Seq ID No:5 from human12q21-22.

Seq ID No:7 corresponds to a cDNA detected by cDNA cloning,corresponding to an mRNA transcript. It includes three exons, the firstand third are identical to the two exons in Seq ID No:6. It istranscribed from the +strand of Seq ID No:1 and Seq ID No:5.doc fromhuman 12q21-22.

Seq ID No:8 corresponds to a cDNA detected by cDNA cloning,corresponding to an mRNA transcript. It includes two exons, the last isalso present as exon-2 in a human cDNA clone (sequence acc. no.BC036936) (Seq ID No:9.doc). It is transcribed from the +strand of SeqID No:5.doc from human 12q21-22.

Seq ID No:9 corresponds to a human cDNA sequence (sequence acc. no.BC036936). It is transcribed from the +strand Seq ID No:5 from 12q21-22.We have not cDNA cloned this cDNA, but a splice variant (Seq ID No:8),where exon-2 of this sequence was spliced to exon-1 of Seq ID No:6 wasdetected by cDNA cloning. It is transcribed from the +strand of Seq IDNo:5 from human 12q21-22.

Seq ID No:10 corresponds to a cDNA detected partly by cDNA cloning,partly by PCR analysis, corresponding to an mRNA transcript. It includestwo exons, exon-1 includes the region encoding Seq ID No:3 and exon-1from Seq ID No:6; exon-2 is identical to exon-2 in Seq ID No:6 andexon-3 in Seq ID No:7. It is transcribed from the +strand of Seq IDNo:1.doc and Seq ID No:5 from human 12q21-22.

Seq ID No:11 corresponds to a cDNA detected by cDNA cloning,corresponding to an mRNA transcript. It includes one exon. The sequenceincludes the region that encodes Seq ID No:3, exons 2 and 3 from Seq IDNo:7 and the region between those exons. It is transcribed from the+strand of Seq ID No:1 and Seq ID No:5 from human 12q21-22.

Seq ID No:12 Is an exon sequence. It corresponds to the first exon inSeq ID No: 9 It is transcribed from the +strand of Seq ID No:5 fromhuman 12q2-22.

Seq ID No:13 Is an exon sequence. It corresponds to the first exon inSeq ID No: 6, 7, 8, and it is included in Seq ID No: 2. It istranscribed from the +strand of Seq ID No: 1 and 5 from human 12q21-22.

Seq ID No:14 Is an exon sequence. It is identical to Seq ID No: 13, butwith an additional GT dinucleotide at the 3′end, caused by the use of analternative splice site. It can replace Seq ID No: 13 as the first exonin Seq ID No: 6, 7, 8, and be included in Seq ID No: 2. It istranscribed from the +strand of Seq ID No:1 and 5 from human 12q21-22.

Seq ID No:15 Is an exon sequence. It corresponds to the second exon inSeq ID No:7 and it is included in Seq ID No: 2, 4, 10 and 11. It istranscribed from the +strand of Seq ID No:1 and 5 from human 12q21-22.

Seq ID No:16 Is an exon sequence. It corresponds to the third exon inSeq ID No:7, it is the second exon in Seq ID No:6 and 11 and it isincluded in Seq ID No:2 and 4. It is transcribed from the +strand of SeqID No:5 from human 12q21-22.

Seq ID No:17 Is the sequence encoding the peptide in Seq ID No: 3. It isincluded in Seq ID No:2, 4, 10 and 11. It is transcribed from the+strand of Seq ID No:5 from human 12q21-22.

Seq ID No:18 Is an exon sequence. It corresponds to the second exon inSeq ID No:8 and 9. It is transcribed from the +strand of Seq ID No:5from human 12q21-22.

Methods of Diagnosis

One important aspect of the present invention relates to diagnosis of asubtype of B-cell chronic lymphocytic leukaemia (B-CLL) having poorprognosis. A further important aspect of the invention relates toprognosis of B-CLL. These methods are based on the discovery by thepresent inventors that an expression product which comprise at least onenucleotide sequence selected from the group consisting of SEQ ID No:12,SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 andSEQ ID No:18 is (are) only present in particular subtypes of B-CLLassociated with poor prognosis and completely absent in other subtypesof B-CLL and in healthy tissue (see in particular example 2). Bycompletely absent is meant that the expression products are not detectedin any of the other tissue types with the methods used in the appendedexamples. This is indicative of a complete absence of any transcript ora very low level of transcript in the other tissue types.

The expression product is encoded by SEQ ID No 1 and/or SEQ ID No 5, andthe expression product is selected from the group consisting oftranscriptional products and translational products.

Thus, the present invention relates to a method for detecting thepresence or absence of at least one expression product, wherein the atleast one expression product comprise a nucleotide sequence selectedfrom the group consisting of SEQ ID No:12, SEQ ID No: 13, SEQ ID No:14,SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 in abiological sample isolated from an individual for establishing adifferential diagnosis of B-CLL or for determining the prognosis of theB-CLL.

“Expression product” is herein meant to be a product which is the resultof the expression of a polynucleotide such as DNA sequence, e.g. agenomic DNA sequence, and is in the form of either a polypeptide or inthe form of a polynucleotide, i.e. an expression product can be selectedfrom the group consisting of a transcriptional product and atranslational product. In the case where the expression product is apolynucleotide, said polynucleotide is preferably mRNA selected from thegroup consisting of mRNA, pre-mRNA, pre-pro-mRNA.

A “transcriptional product” or a “transcription product” is herein meantto be a product resulting from a transcription of a polynucleotide suchas a DNA molecule, preferably a genomic DNA molecule. A transcriptionalproduct is inherently a nucleotide, such as an oligonucleotide or apolynucleotide.

A “translational product” or a “translation product” is herein meant tobe a product resulting from a translation of a transcriptional productsuch as a mRNA. A translational product is inherently a oligopeptide ora polypeptide.

The expression product of the present invention has almost exclusivelybeen found as transcription products in patients with poor B-CLLprognosis. Based on the experimental data presented in the herein, theinventors expect that it turns out that the subtype of B-CLL ischaracterised solely or better by the presence of a transcriptional ortranslational product which comprise a sequence selected from the groupconsisting of SEQ ID No:3, SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18.

Preferably the individual is a mammal, more preferably a human. It isalso expected that the gene encoded by SEQ ID No 1 and/or SEQ ID No 5and the expression products derived from said gene can be used as adiagnostic tool in other species in particular in mammals selected fromthe group: domestic animals such as cow, horse, sheep, pig; and petssuch as cat or dog.

In the case that the expression product is a transcriptional product,this transcriptional product just needs to comprise at least one of thenucleotide sequences selected from the group consisting of SEQ ID No:12,SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 andSEQ ID No:18. These transcriptional products will preferably be derivedfrom SEQ ID No 1 and/or SEQ ID No 5 and may be in the form of mRNA orany pre- or pro-forms of said mRNA. As described, the transcriptionalproduct may comprise at least one of the nucleotide sequences selectedfrom the group consisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14,SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18, such as oneof the nucleotide sequences selected from the group consisting of SEQ IDNo:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ IDNo:17 and SEQ ID No:18, such as two of the nucleotide sequences selectedfrom the group consisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14,SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18, such as 3 ofthe nucleotide sequences selected from the group consisting of SEQ IDNo:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ IDNo:17 and SEQ ID No:18, e.g. 4 of the nucleotide sequences selected fromthe group consisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ IDNo:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18, such as 5 of thenucleotide sequences selected from the group consisting of SEQ ID No:12,SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 andSEQ ID No:18 and ultimately the transcriptional product may comprise allof the six nucleotide sequences selected from the group consisting ofSEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16,SEQ ID No:17 and SEQ ID No:18. The transcriptional product of thepresent invention can have any sequence which is a result of combiningthe nucleotide sequences selected from the group consisting of SEQ IDNo: 12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ IDNo:17 and SEQ ID No:18 as long as the specific nucleotide sequencesselected from the group consisting of SEQ ID No:12, SEQ ID No:13, SEQ IDNo:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 can beidentified as intact sequences in the transcriptional product.

Examples of transcriptional products in the form of specific mRNAs whichcomprise at least one of the nucleotide sequences selected from thegroup consisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ IDNo:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 are the sequencescorresponding to SEQ ID No 2 (short cDNA clone) SEQ ID No 4 (long cDNAclone) SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10and SEQ ID No 11. These mRNA sequences have been found in patients withpoor prognosis.

It is obvious for a person skilled in the art that any fragments of SEQID No:2, SEQ ID No:4, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ IDNo:9, SEQ ID No:10 and SEQ ID No:11 will have the same diagnostic valueas long as the nucleotide sequences selected from the group consistingof SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16,SEQ ID No:17 and SEQ ID No:18 can be found in the fragments.

The mRNA sequence may be detected in a sample using hybridisationtechniques. In particular when more than one analysis is to be performedat the same time it is advantageous to use a DNA array comprising e.g.an oligomer of at least 15 consecutive bases selected from the groupconsisting of SEQ ID No:2, SEQ ID No:4, SEQ ID No:6, SEQ ID No:7, SEQ IDNo:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ IDNo:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ IDNo:18.

Another way of detecting the presence or absence of the transcriptionalproduct is by specifically amplifying the transcriptionals producthaving a sequence corresponding to SEQ ID No 2, 4, 6, 7, 8, 9, 10 or 11or fragments thereof. This can be done by selecting primer pairs whichcause only the amplification of these sequences.

Generally, hybridisation techniques are selected from not limited to thegroup consisting of in situ hybridisation, northern blots, Southernblots, dot blots and PCR based techniques.

A non-limiting list of PCR based techniques include rt-PCR, quantitativePCR and realtime PCR.

According to yet another embodiment, the translational product is aprotein encoded by a polynucleotide selected from the group consistingof SEQ ID No:1, SEQ ID No:5, SEQ ID No:2, SEQ ID No:4, SEQ ID No:6, SEQID No:7, SEQ ID No:8, SEQ ID No:9, SEQ ID No:10 and SEQ ID No:11.Detection of this protein can be done with state of the art methodsincluding the detection with an antibody directed against said protein,such as Western blotting, more preferably by using a fluorescentlylabelled antibody, preferably wherein the method comprises the use offlowcytometry, such as FACS. Other methods include but are not limitedto gel electrophoresis, gel filtration, ion exchange chromatography,FPLC, mass spectrometry and immunohistochemistry.

Preferably, said protein is selected from the group comprising SEQ ID No3 (protein), or a protein sharing at least 60% sequence identity withSEQ ID No 3. The protein with the amino acid sequence set forth in SEQID No 3 is the longest open reading frame in the cDNA sequence of SEQ IDNo 2 or 4.

In a specific embodiment of the present invention is a method fordetermining whether an individual has a B-CLL sub-type with poorprognosis, the method comprising determining the level of an expressionproduct which comprise a nucleotide sequence selected from the groupconsisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15,SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 of said individual, andindicating the individual as having a B-CLL sub-type with poor prognosisif the level of the expression product is at or beyond a discriminatingvalue and indicating the individual as not having a B-CLL sub-type withpoor prognosis if the level of the expression product is not at orbeyond the discriminating value, the discriminating value being a valuewhich has been determined by measuring the level of the expressionproduct which comprise a nucleotide sequence selected from the groupconsisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15,SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 in both a healthy controlpopulation and a population with known B-CLL sub-type with poorprognosis, thereby determining said discriminating value whichidentifies the B-CLL sub-type population having a poor prognosis.

In this method the individual may be a member of an unselectedpopulation or be a member of a population already identified as having aB-CLL sub-type with a poor prognosis.

The above method may be performed such that the determination isperformed at several time points at intervals as part of a monitoring ofa cancer patient after or during the treatment for primary cancer.

The methods described so-far relate to the determination of the presenceor absence of an expression product which comprise a nucleotide sequenceselected from the group consisting of SEQ ID No:12, SEQ ID No:13, SEQ IDNo:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18. Byquantitatively measuring the amount of an expression product whichcomprise a nucleotide sequence selected from the group consisting of SEQID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ IDNo:17 and SEQ ID No:18 in a biological sample isolated from anindividual, it is possible to predict the progression of B-CLL in anindividual.

In one embodiment the quantitative measurement is performed duringtreatment to estimate the efficiency of such treatment.

A preferred embodiment of the above diagnostic and prognostic methods isa method for detecting the presence or absence an expression product,wherein said at least one expression product comprise the nucleotidesequence of SEQ ID No:15 in a biological sample isolated from anindividual for establishing a differential diagnosis of B-CLL or fordetermining the prognosis of the B-CLL.

A further preferred embodiment of the above diagnostic and prognosticmethods is a method for detecting the presence or absence an expressionproduct, wherein said at least one expression product comprise thenucleotide sequence of SEQ ID No:16 in a biological sample isolated froman individual for establishing a differential diagnosis of B-CLL or fordetermining the prognosis of the B-CLL.

Yet a further preferred embodiment of the above diagnostic andprognostic methods is a method for detecting the presence or absence anexpression product, wherein said at least one expression productcomprise the nucleotide sequence spanning the junction sequence betweenExon-2 (SEQ ID No:15) and Exon-3 (SEQ ID No:16) in a biological sampleisolated from an individual for establishing a differential diagnosis ofB-CLL or for determining the prognosis of the B-CLL.

The nucleotide sequence spanning the junction between Exon-2 and Exon-3is the last 20 nucleotides of the 3′-end of SEQ ID No: 15 and the first20 nucleotides of the 5′-end of SEQ ID No:16.

The “junction sequence” between two nucleotide sequences, such as twoexons, is herein defined as the at least 20 3′-nucleotides of the firstexon which is located 5′ relative to the second exon and the at least 205′-nucleotides of the second exon which is located 3′ relative to thefirst exon.

For all diagnostic applications of the present invention, the biologicalsample may be selected from the group comprising blood, serum, plasma,urine, saliva, lymph node biopsy, bone marrow, spinal liquid, spleenbiopsy, and liver biopsy. The cells to be assessed in a sample arepreferably leukocytes, mononuclear leukocytes or lymphocytes orB-lymphocytes.

A further embodiment of the present invention also includes a diagnostickit for ex vivo or in situ diagnosis of a subtype of B-cell chroniclymphocytic leukaemia (B-CLL) in a individual, the kit comprising adetector molecule capable of detecting the presence or absence of atleast one expression product, wherein said at least one expressionproduct comprise a nucleotide sequence selected from the groupconsisting of SEQ ID SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ IDNo:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18 in a biologicalsample isolated from the individual.

The detector molecule is preferably a nucleotide and even morepreferably a nucleotide capable of hybridising to a nucleotide sequenceselected from the group consisting of SEQ ID SEQ ID No:12, SEQ ID No:13,SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18under stringent condition.

B-CLL Therapy

With the identification of a new sub-type of B-CLL having a poorprognosis, the present inventors also provide methods for treatment ofB-CLL in such patients. This method is based on the finding thattranscription products comprising these sequence products are present inB-CLL cells of patients with the poor prognosis. By modifying theactivity and/or level of these transcription products, a treatmentand/or cure for B-CLL is provided.

Accordingly, in a therapeutic aspect of the present invention there isprovided a method of treating a B-CLL sub-type with poor prognosiscomprising administering to an individual with a poor prognosis B-CLLdiagnosis a compound capable of decreasing or inhibiting the formationof an expression product which comprise a nucleotide sequence selectedfrom the group consisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14,SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18.

One such method is based on administering an oligonucleotide capable ofinhibiting transcription from SEQ ID No 1 and/or SEQ ID No 5. Saidoligonucleotide may comprises at least 8-10 consecutive nucleotides fromthe sequence of SEQ ID No 1. These sequences constitute the putativepromoter sequences controlling the transcription products which comprisea nucleotide sequence selected from the group consisting of SEQ IDNo:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ IDNo:17 and SEQ ID No:18. The oligonucleotides bind specifically to thepromoter sequences and inhibit transcription of the gene. Sucholigonucleotides may comprises nucleotide monomers selected from thegroup: DNA, RNA, LNA, PNA, methylated DNA, methylated RNA, morepreferably PNA or LNA.

In a more preferred embodiment the therapeutic methods compriseadministering an oligonucleotide capable of binding to a transcriptionalproduct and preventing translation by destroying the transcriptionalproduct. One particularly preferred embodiment of this aspect is RNAinterference (RNAi) oligonucleotides.

The discovery of the phenomenon RNAi has revealed an entirely new levelof gene regulation in eukaryotic cells. It is based on the observationthat the presence of long double stranded RNA (dsRNA) in a cell almostcompletely eliminates the expression of the gene having the samesequence, whereas expression of other unrelated genes are leftundisturbed. Although this observation had been know for time in plantsas posttranscriptional gene silencing (PTGS) it was not before it wascharacterised as a general mechanism throughout the animal kingdom thatits potentials were fully appreciated. Over the last few years it hasbeen developed as a robust technique to knock down any desirable gene inworms and flies, and quickly a large body of information was gatheredabout the function of genes in these organisms. Due to the activation ofthe interferon system by long dsRNA the RNAi method was at that time notapplicable in a mammalian system.

A key observation that allowed the harnessing of RNAi as a tool forregulating gene expression in mammals was the observation thatchemically synthesised oligo-mer small interfering RNAs (siRNA)effectively suppress gene expression in several human cell lines withoutinflecting interferon response. This has triggered new promises forsiRNA as a therapeutic drug in humans.

RNAi works by hybridising specifically to the mRNA transcribed by thecell to form a (partly) double stranded RNA molecule. This is recognisedas a double stranded molecule by the cell's own nucleases, which degradethem.

In order for the technique to work efficiently, the siRNAoligonucleotide comprises a sequence of 5-30 consecutive nucleotideswhich is the complementary sequence of the nucleotide sequences selectedfrom the group consisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14,SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18. By targetingat least one of the nucleotide sequences selected from the groupconsisting of SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No: 15,SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18, the transcriptionalproducts characterised by the nucleotide sequences from the group of SEQID No:2, SEQ ID No:4, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ IDNo:9, SEQ ID No:10 and SEQ ID No:11 will be eliminated. Example 5 showsthat cells characteristic for the poor prognosis B-CLL sub-type can beeliminated by destroying the herein mentioned transcription products.

By “complementarity” is meant a nucleic acid that can form hydrogenbond(s) with other DNA or RNA sequence by either traditionalWatson-Crick or other non-traditional types of base-paired interactions,e.g. Hoogsteen type.

Preferred siRNA molecules of the present invention are between 5 to 30nucleotides long, such as 8-30 nucleotides long, such as 8-25nucleotides, e.g. 8-24 nucleotides, e.g. 8-23 nucleotides, e.g. 8-22nucleotides, e.g. 8-21 nucleotides, such as 8-20 nucleotides, e.g. 9-23nucleotides, e.g. 10-23 nucleotides, such as 11-23 nucleotides, e.g.12-23 nucleotides such as 13-23 nucleotides, e.g. 14-23 nucleotides,e.g. 15-23 nucleotides, such as 16-23 nucleotides, such as 8nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28nucleotides, 29 nucleotides, such as 30 nucleotides long.

RNAi oligonucleotides may be administered to the cell, or a vector maybe transfected into the cells, said vector comprising a promoter regioncapable of directing the expression of at least one RNAioligonucleotide. Due to the very restricted expression of the AMB-1gene, it is not important only to target the RNAi oligos or the vectorsto B-CLL cells.

One way of targeting to blood cells comprises using a heparin receptorfor targeting to blood cells.

Another way of targeting the transcriptional products which comprise atleast one nucleotide sequence selected from the group consisting of SEQID No:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ IDNo:17 and SEQ ID No:18 is to use an antisense construct comprising apromoter sequence capable of directing the transcription of at leastpart of the antisense equivalent of SEQ ID No 1 or 2 or 4 or 5 or 6 or 7or 8 or 9 or 10 or 11 specifically to the poor prognosis B-CLL sub-type.

When desired targeting to B-CLL cells can be performed using the CD19 orCD20 receptor. The CD19 receptor is particularly preferred since itinternalises its ligand.

In a further therapeutic embodiment the compound is a gene therapyvector comprising a promoter sequence operably linked to a sequencecoding for a protein capable of inhibiting cell division in the celland/or capable of killing the cell, said promoter sequence being atissue specific promoter capable of directing expression only in Bcells, more preferably only in B-CLL cells. One particularly preferredpromoter sequence is the extremely cell specific promoter of SEQ ID No:1or SEQ ID No:5. When this promoter is used targeting of the suicidevector is not very important, since it will only be active in the cellsin which AMB-1 is expressed and these are the cells to be targeted bythe suicide gene.

Deletion studies will determine the exact length of the promotersequence counted from the transcription start site. Accordingly, thepromoter may comprise at least 100 nucleotides of Seq_ID:1 or Seq_ID:5,such as at least 200 nucleotides, for example at least 300 nucleotides,such as at least 400 nucleotides, for example at least 500 nucleotides,such as at least 600 nucleotides, for example at least 700 nucleotides,such as at least 800 nucleotides, for example at least 900 nucleotides,such as at least 1000 nucleotides, for example at least 1100nucleotides, such as at least 1200 nucleotides, for example at least1300 nucleotides, such as at least 1400 nucleotides, for example atleast 1500 nucleotides, such as at least 1600 nucleotides, for exampleat least 1700 nucleotides, such as at least 1800 nucleotides, forexample at least 1900 nucleotides, such as at least 2000 nucleotides,for example at least 2500 nucleotides, such as at least 3000nucleotides, for example at least 3500 nucleotides, such as at least5000 nucleotides, for example at least 10,000 nucleotides.

The specificity of expression of mRNAs described by the presentinvention are striking. The RT-PCR data and the Northern blot data usingthe dot-blot disclose that the mRNAs of the present invention areexpressed either at very low levels in other tissues or only in theB-CLL patients where one can detect it.

Thus one embodiment of the present invention is the use of the promotorregion for use in gene therapy. The promotor is defined as any sequencewithin SEQ ID No:1 and SEQ ID No:5 that directs the formation of anexpression product which comprise a nucleotide sequence selected fromthe group consisting of SEQ ID No: 12, SEQ ID No:13, SEQ ID No:14, SEQID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ ID No:18, said expressionproduct being any of the sequences selected from the group consisting ofSEQ ID No:2, SEQ ID No:4, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ IDNo:9, SEQ ID No:10 and SEQ ID No:11 in B-CLL cells or any other cell ortissue types in which any of the sequences are transcribed.

One way that one can use the promotor region in gene therapy is to makea gene therapy construct where the promotor drives the expression of acell suicide gene such as but not limited to the gene for HSV-1thymidine kinase, the varicella-zoster, virus thymidine kinase gene, E.Coli cytosine deaminase, the nitroreductase gene or the E. Coli Deo gene(Yazawa et al. & Kirn D. et al.). This would allow for selectiveexpression of the suicide genes in B-CLL cells.

Alternatively, the promotor could be used for a selective expression ofgenes that could have curative effects when expressed in B-CLL cells,but unwanted effects if expressed ubiquitously.

Also, one embodiment of the present invention relates to the use of thepromotor region for use in screening assays where the promotor is linkedto a reporter gene and transfected into B-CLL cells in which thereporter gene will be expressed. This approach would allow for easyscreening for compounds that would turn off the expression of thereporter gene for example by killing the cell.

A presently preferred embodiment relates a gene therapy vector of thepresent invention comprising an oligonucleotide capable of inhibitingtranscription from SEQ ID No 1 and/or SEQ ID No 5, wherein the promoteris a B-CLL specific promoter, which may or may not be operably linked toa protein selected from the group comprising HSV-1 thymidine kinase, thevaricella-zoster, virus thymidine kinase gene, E. Coli cytosinedeaminase, the nitroreductase gene or the E. Coli Deo gene.

In one embodiment the compound is a therapeutic antibody directedagainst a polypeptide having the amino acid sequence of SEQ ID No 3,preferably wherein said antibody is a human or humanised antibody.Another possibility is to identify a modulator of binding of SEQ ID No 3to its receptor within or outside the cell and to administer thismodulator to the cells.

4-Helical Cytokines

A further object of preferred embodiments of the present invention is anisolated polypeptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   i) an amino acid sequence of SEQ ID NO: 3,    -   ii) an amino acid sequence having at least 60% sequence identity        compared to the full length sequence of SEQ ID NO:3    -   ii) a fragment of SEQ ID NO:3 having at least 60% sequence        identity compared to the full length sequence of SEQ ID NO:3.

The protein encoded by SEQ ID No 1 and/or SEQ ID No 5 shares a verysmall sequence identity with any known protein. However, it has beenpossible to use 2D and 3D analytical tools to Identify the protein as a4-helical cytokine. The 3D structure of the protein is very similar to4-helical cytokines and in particular to IL4.

IL4 is a very important cytokine in B-CLL biology. IL4 is not expressedby B-CLL cells, but the IL4 receptor is found on the cells. The IL4 thatstimulates B-CLL cells is believed to be produced by T-lymphocytes. Therole of IL4 in B-CLL biology is complicated. It has been suggested thatIL4 can inhibit B-CLL DNA synthesis and proliferation. Other reportsdemonstrated that IL4 protects B-CLL cells from apoptosis byupregulating Bcl-2, and IL4 was shown to inhibit apoptosis withoutstimulating proliferation. Recently, a clinical study in Sweden hasconfirmed these in vitro studies since IL4 administration to B-CLLpatients resulted in increased numbers of B-CLL cells in the blood,suggesting that IL4 had a stimulatory or anti-apoptotic effect on theB-CLL cells in vivo (Lundin, et al.).

In many systems the effects of IL13 are largely similar to those of IL4,but IL13 is slightly less potent that IL4. It is unclear whether B-CLLcells express IL13, but the cells do express the IL13 receptor. Theeffects of IL13 in B-CLL are controversial. While Chaouchi et al.suggested that IL13, like IL4 protects B-CLL cells from apoptosis(Chaouchi et al), studies by Fluckiger et al. suggest that this is notthe case (Fluckiger et al.).

The combined finding of 2D and 3D structure similarity to 4-helicalcytokines and the importance of IL4 in B-CLL strongly suggests that thenovel class of proteins of which the AMB-1 protein is one representativeare cytokines.

These polypeptides constitute a novel class of proteins sharing 2D and3D structure similarities with 4-helical cytokines. In a preferredembodiment, the isolated polypeptide comprises or essentially consistsof the amino acid sequence of SEQ ID No. 3 or a fragment thereof. Thisparticular protein at least can be used for diagnosis, for raisingantibodies for use in therapy against B-CLL, and for protective ortherapeutic immunisation of an individual against B-CLL.

Consequently, the isolated polypeptide preferably has interleukinactivity or interleukin like activity, such as having IL3, IL13, GM-CSF,TGF-β, IGF activity, more preferably having IL4 activity or IL4 likeactivity.

Probably the isolated polypeptides are capable of forming homo- orhetero-oligomer with each other and among themselves. Such oligomers arealso within the scope of the present invention. Such oligomers maycomprise at least one isolated polypeptides as defined in any thepresent invention, such as a dimer, a trimer, a quatramer, a quintamer,a hexamer, an octamer, a decamer, a dodecamer. In biological systems theactivity may be attributed only to dimer or higher-mer.

The protein defined by SEQ ID No 3 shares very little sequence identitywith known cytokines and interleukines and as a matter of fact verylittle sequence identity with any known protein. Consequently thepresent inventors contemplates that the group comprises functionallyequivalent polypeptide sharing at least 60% sequence identity with SEQID No 3, more preferably at least 70% sequence identity, more preferablyat least 80% sequence identity, such as at least 90% sequence identity,for example at least 95% sequence identity, such as at least 97%sequence identity, for example at least 98% sequence identity.

Activity as a cytokine or interleukin can be assessed in a biologicalassay where the polypeptide is contacted with a cytokine dependent cellline. Accordingly, polypeptides with cytokine or interleukin likeactivity can also be identified by similar methods.

One approach to assess cytokine/interleukin activity in a biologicalassay is to express the CDS (SEQ ID No:17) reading frame in abaculovirus system (Invitrogen, Carlsbad, USA) and purify the protein.The recombinant protein can be assayed in cytokine induced proliferationassays as described in general in the eBioscience catalog & ReferenceManual 2002 p. 260-262 (eBioscience, San Diego, USA). In particular IL4activity can be determined using the CTh4S cell line as described byPetersen et al (see Example 8).

The promoter sequence (which forms part of SEQ ID No 1 and/or SEQ ID No5) and the coding sequences (SEQ ID No:3) can be used in various aspectsof gene therapy and immunotherapy.

Further polynucleotide sequences from other individuals or other specieswith the same function can be isolated by one of the following methods,which each form independent aspects of the present invention.

A first method for identifying a nucleotide sequence encoding a4-helical cytokine comprises the steps of:

i) isolating mRNA from a biological sample,ii) hybridising the mRNA to a probe comprising at least 10 nucleotidesof the coding sequence of SEQ ID No 1 and/or SEQ ID No 5 under stringentconditions,iii) determining the nucleotide sequence of a sequence capable ofhybridising under step ii), andiv) determining the presence of an open reading frame in the nucleotidesequence determined under step iii).

A second method for identifying a nucleotide sequence encoding a4-helical cytokine is a computer assisted method comprising the steps of

i) performing a sequence similarity search of at least 10 nucleotides ofthe coding sequence SEQ ID No 1 and/or SEQ ID No 5,ii) aligning “hits” to said coding sequence,iii) determining the presence of an open reading frame in the “hits”.

It is highly likely that other similar polypeptides encoding further4-helical cytokines can be found in other individuals and/or otherspecies of mammals. In particular, individuals of other geographicalorigin may carry genes which differ from the polynucleotides of thepresent invention. It is also conceivable that similar sequences can befound in closely and even in distantly related species.

Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thegenetic code.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies, binding sites of receptors, or binding sites onsubstrate molecules. Since it is the interactive capacity and nature ofa protein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence, and, of course, its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in thepeptide sequences of the disclosed compositions, or corresponding DNAsequences which encode said peptides without appreciable loss of theirbiological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics(Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, ie. still obtain abiological functionally equivalent protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred. It is alsounderstood in the art that the substitution of like amino acids can bemade effectively on the basis of hydrophilicity. U.S. Pat. No.4,554,101, incorporated herein by reference, states that the greatestlocal average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Functional equivalents and variants are used interchangably herein. Inone preferred embodiment of the invention there is also providedvariants of a 4-helical cytokine, and variants of fragments thereof.When being polypeptides, variants are determined on the basis of theirdegree of identity or their homology with a predetermined amino acidsequence, said predetermined amino acid sequence being SEQ ID No. 3 or afragment thereof.

Accordingly, variants preferably have at least 60% sequence identity,for example at least 65% sequence identity, such as at least 70%sequence identity, for example at least 75% sequence identity, forexample at least 80% sequence identity, such as at least 85% sequenceidentity, for example at least 90% sequence identity, such as at least91% sequence identity, for example at least 91% sequence identity, suchas at least 92% sequence identity, for example at least 93% sequenceidentity, such as at least 94% sequence identity, for example at least95% sequence identity, such as at least 96% sequence identity, forexample at least 97% sequence identity, such as at least 98% sequenceidentity, for example 99% sequence identity with the predeterminedsequence.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity # ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength. The sequence identity is preferably calculated relative to thefull length sequence of the molecule of the present invention.

A degree of “sequence identity” of amino acid sequences is a function ofthe number of identical amino acids at positions shared by the aminoacid sequences. A degree of “sequence homology” or “sequence similarity”of amino acid sequences is a function of the number of amino acids, i.e.structurally related, at positions shared by the amino acid sequences.Sequence identity is determined by the alignment algorithm that performsglobal alignments which has been described by Smith T F and Waterman M S(Smith T F et al.)

A list of the standard qualifiers and the default values for thealignment algorithm is given below:

Allowed values Default Standard (Mandatory) qualifiers [-asequence]Sequence USA Readable sequence Required (Parameter 1) [-bsequence]Sequence database USA Readable Required (Parameter 2) sequence(s)-gapopen The gap open penalty is the score taken Number from 1.000 10.0for any away when a gap is created. The best value to 100.000 sequencedepends on the choice of comparison matrix. The default value assumesyou are using the EBLOSUM62 matrix for protein sequences, and theEDNAFULL matrix for nucleotide sequences. -gapextend The gap extensionpenalty is added to the Number from 0.100 0.5 for any standard gappenalty for each base or to 10.000 sequence residue in the gap. This ishow long gaps are penalized. Usually you will expect a few long gapsrather than many short gaps, so the gap extension penalty should belower than the gap penalty. An exception is where one or both sequencesare single reads with possible sequencing errors in which case you wouldexpect many single base gaps. You can get this result by setting the gapopen penalty to zero (or very low) and using the gap extension penaltyto control gap scoring. [-outfile] Output alignment file name Alignmentoutput (Parameter 3) file Additional (Optional) qualifiers -data fileThis is the scoring matrix file used when Comparison matrix EBLOSUM62for comparing sequences. By default it is the file in EMBOSS dataprotein EDNAFULL file ‘EBLOSUM62’ (for proteins) or the file path forDNA ‘EDNAFULL’ (for nucleic sequences). These files are found in the‘data’ directory of the EMBOSS installation. Advanced (Unprompted)qualifiers -[no]brief Brief identity and similarity Boolean value YesYes/No

An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25% identity, with one of the4-helical cytokine sequences of the present invention. The term“substantial identity” means that two peptide sequences, when optimallyaligned, such as by the Smith and Waterman algorithm using default gapweights, share at least 80 percent sequence identity, preferably atleast 90 percent sequence identity, more preferably at least 95 percentsequence identity or more (e.g., 99 percent sequence identity).Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Additionally, variants are also determined based on a predeterminednumber of conservative amino acid substitutions as defined herein below.Conservative amino acid substitution as used herein relates to thesubstitution of one amino acid (within a predetermined group of aminoacids) for another amino acid (within the same group), wherein the aminoacids exhibit similar or substantially similar characteristics.

Within the meaning of the term “conservative amino acid substitution” asapplied herein, one amino acid may be substituted for another within thegroups of amino acids indicated herein below:

-   -   Amino acids having polar side chains (Asp, Glu, Lys, Arg, His,        Asn, Gin, Ser, Thr, Tyr, and Cys,)    -   Amino acids having non-polar side chains (Gly, Ala, Val, Leu,        Ile, Phe, Trp, Pro, and Met)    -   Amino acids having aliphatic side chains (Gly, Ala Val, Leu,        Ile)    -   Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)    -   Amino acids having aromatic side chains (Phe, Tyr, Trp)    -   Amino acids having acidic side chains (Asp, Glu)    -   Amino acids having basic side chains (Lys, Arg, His)    -   Amino acids having amide side chains (Asn, Gin)    -   Amino acids having hydroxy side chains (Ser, Thr)    -   Amino acids having sulphor-containing side chains (Cys, Met),    -   Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser,        Thr)    -   Hydrophilic, acidic amino acids (Gin, Asn, Glu, Asp), and    -   Hydrophobic amino acids (Leu, Ile, Val)

Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Accordingly, a variant or a fragment thereof according to the inventionmay comprise, within the same variant of the sequence or fragmentsthereof, or among different variants of the sequence or fragmentsthereof, at least one substitution, such as a plurality of substitutionsintroduced independently of one another.

It is clear from the above outline that the same variant or fragmentthereof may comprise more than one conservative amino acid substitutionfrom more than one group of conservative amino acids as defined hereinabove.

The addition or deletion of at least one amino acid may be an additionor deletion of from preferably 2 to 250 amino acids, such as from 10 to20 amino acids, for example from 20 to 30 amino acids, such as from 40to 50 amino acids. However, additions or deletions of more than 50 aminoacids, such as additions from 50 to 100 amino acids, addition of 100 to150 amino acids, addition of 150-250 amino acids, are also comprisedwithin the present invention. The deletion and/or the additionmay—independently of one another—be a deletion and/or an addition withina sequence and/or at the end of a sequence.

The polypeptide fragments according to the present invention, includingany functional equivalents thereof, may in one embodiment comprise lessthan 250 amino acid residues, such as less than 240 amino acid residues,for example less than 225 amino acid residues, such as less than 200amino acid residues, for example less than 180 amino acid residues, suchas less than 160 amino acid residues, for example less than 150 aminoacid residues, such as less than 140 amino acid residues, for exampleless than 130 amino acid residues, such as less than 120 amino acidresidues, for example less than 110 amino acid residues, such as lessthan 100 amino acid residues, for example less than 90 amino acidresidues, such as less than 85 amino acid residues, for example lessthan 80 amino acid residues, such as less than 75 amino acid residues,for example less than 70 amino acid residues, such as less than 65 aminoacid residues, for example less than 60 amino acid residues, such asless than 55 amino acid residues, for example less than 50 amino acidresidues.

“Functional equivalency” as used in the present invention is accordingto one preferred embodiment established by means of reference to thecorresponding functionality of a predetermined fragment of the sequence.

Functional equivalents or variants of a 4-helical cytokine will beunderstood to exhibit amino acid sequences gradually differing from thepreferred predetermined 4-helical cytokine, as the number and scope ofinsertions, deletions and substitutions including conservativesubstitutions increases. This difference is measured as a reduction inhomology between the preferred predetermined sequence and the fragmentor functional equivalent.

All fragments or functional equivalents of SEQ ID No. 3 are includedwithin the scope of this invention, regardless of the degree of homologythat they show to the respective, predetermined 4-helical cytokinesdisclosed herein. The reason for this is that some regions of the4-helical cytokines are most likely readily mutatable, or capable ofbeing completely deleted, without any significant effect on the bindingactivity of the resulting fragment.

A functional variant obtained by substitution may well exhibit some formor degree of native cytokine activity, and yet be less homologous, ifresidues containing functionally similar amino acid side chains aresubstituted. Functionally similar in this respect refers to dominantcharacteristics of the side chains such as hydrophobic, basic, neutralor acidic, or the presence or absence of steric bulk. Accordingly, inone embodiment of the invention, the degree of identity is not aprincipal measure of a fragment being a variant or functional equivalentof a preferred predetermined fragment according to the presentinvention.

One particularly preferred method of determining the degree offunctional equivalence is by performing a biological or chemical assaysuch as the assays described in the appended examples. Preferredfunctional equivalents of SEQ ID No 3 are those that have a K_(D) withrespect to a predefined receptor which is less than 10 times higher thanthe K_(D) of the polypeptide of SEQ ID No 1 with respect to the samereceptor, more preferably less than 5 times higher, more preferably lessthan 2 times higher.

With respect to functional equivalence this may be defined in abiological assay based on a cytokine dependent or stimulated cell line.Such cell lines are e.g. available from American Type CultureCollection, P.O. Box 1549, Manassas, Va. 20108 USA. The following celllines at least are available for testing cytokines and in particularinterleukins:

Accession number Description Activity CRL-1841 TH-2 clone A5E IL2dependent, IL4 stimulated CRL-2003 TF-1 IL3 dependent CRL-2407 NK-92 IL2dependent CRL-2408 NK-92MI IL2 dependent CRL-2409 NK92CI IL2 dependentCRL-9589 AML-193 IL3 stimulated, GM-CSF dep. CRL-9591 MV-4-11 GM-CSFdependent TIB-214 CTLL-2 IL2 dependent

The following cell lines are available from DSMZ—Deutsche Sammiung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124Braunschweig, GERMANY. As can be seen from the table, some of the celllines can be used to broadly assess cytokine activity whereas others areonly reported to respond to one or a few specific cytokines.

Accession number Description Acvitity ACC 211 Mouse hybridoma, B9 IL6dependent ACC 137 Human acute myeloid leukemia, UT-7 Constitutivelycytokine responsive to various cytokines. ACC 104 Human acutemegakaryoblastic leukemia Respond with proliferation to: GM-CSF,IFN-alpha, IFN-á, IFN-gamma, IL2, IL3, IL4, IL6, IL15, NG F, SCF,TNF-alpha, TPO ACC 247 Human acute myeloid leukemia, OCI- G-GSF, GM-CSF,IL3, FTL3- AML5 ligand ACC 271 Human acute myeloid leukemia, MUTZ-2 IL3,SCF, G-CSF, M-CSF, IFN-gamma ACC 334 Human erythroleukemia, TF-1 GM-CSF,IFN-gamma, IL3, IL4, IL5, IL6, IL13, LIF, NGF, OSM, SCF, TNF- alpha, andTPO

The TF-1 cell line mentioned above can be used for assaying IL13function. This cell line is sensitive to various different cytokines butgives a very strong proliferative response when exposed to IL13. Thiscell line can in particular be used if there is no response in the IL4sensitive cell line (CT.h4S). Further cell lines which can be used fordistinguishing between IL4 and IL13 activity include celllines/hybridomas such as B-9-1-3 (Bouteiller et al.) and A201.1 (Andrewset al.).

Pharmaceutical Uses of Isolated Polypeptides

Apart from being used for diagnosis, it is also within the scope of thepresent invention to use an isolated polypeptide as defined in theinvention for a pharmaceutical composition together with apharmaceutically acceptable carrier. Such pharmaceutical compositionsmay be used for any of the purposes for which cytokines and inparticular interleukin is used at present.

Examples of such uses include the treatment of bone disorders,inflammation, for lowering blood serum cholesterol, allergy, infection,viral infections, hematopoietic disorders, preneoplastic lesions, immunerelated diseases, autoimmune related diseases, infectious diseases,tuberculosis, cancer, viral diseases, septic shock, reconstitution ofthe haematopoietic system, induction of the granulocyte system, pain,cardial dysfunction, CNS disorders, depression, artheritis, psoriasis,dermatitis, collitis, Crohn's disease, diabetes, in an individual inneed thereof.

It is also within the scope of the present invention to use an isolatedpolypeptide according to the invention as an adjuvant or as an immuneanhancer, for regulating TH2 immune responses, and for suppressing Th1immune responses.

A further use of an isolated polypeptide of the invention is as a growthfactor for administration to cell cultures or as a growth factor forveterinary use, e.g. for stimulating the growth of livestock.

Immunotherapy

Having identified a transcriptional and/or translational product of SEQID No 1 and/or SEQ ID No 5 as an etiological factor in B-CLL it is alsowithin the scope of the present invention to perform an immunisation ofa patient in need thereof against B-CLL, wherein the immunisationgenerates an immune response in the patient which recognises atranslational product of SEQ ID No 2, SEQ ID No 4, SEQ ID No 6, SEQ IDNo 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10 and SEQ ID No 11. Apreferred immunotherapy is a vaccination against B-CLL by immunising anindividual against a translational product of SEQ ID No 1 and/or SEQ IDNo 5. In this way the individual builds up antibodies directed againstsaid translational product and any developing B-CLL will be stopped bythese antibodies.

Immunisation may be performed in various ways, such as by immunisingsaid individual with at least one isolated polypeptide as defined thepresent invention and optionally adjuvants and carriers or immunisingwith an expression construct capable of expressing an isolatedpolypeptide according to the invention in the cells (DNA vaccination).

Another method comprises peptide loading of dendritic cells, or ex vivoexpansion and activation of T-cells, or inducing a CTL response thattargets cells expressing the polypeptide encoded by SEQ ID No 1 and/orSEQ ID No 5.

Antibodies

Antibodies against any of the polypeptides belonging to the novel classof proteins identified by the present inventors can be produced by anyknown method of immunisation.

In one embodiment, the antibodies are produced in a non-human mammal, orin an insect. If antibodies are to be used for therapy in human beingsthey are preferably subsequently humanised. In one embodiment, theantibody is formulated into a single-chain antibody.

In another embodiment, in particular for therapeutic purposes, the hostorganism is a human being and the antibody is subsequently producedrecombinantly in a non-human mammal, such as a mouse. The antibody mayalso be produced as a monoclonal antibody in a hybridoma. One way ofproducing a monoclonal antibody is described in U.S. Pat. No. 5,681,729in which a human lymphocyte producing an antibody is generated by thesteps, in the order mentioned, comprising

-   -   1. transplanting human lymphocytes to a mouse lacking both        functional T and B cells so that said human lymphocytes take in        said mouse's body;    -   2. immunizing said mouse with a desired antigen so as to        generate human lymphocytes producing an antibody specific to        said antigen;    -   3. administering to said mouse an antiserum to mouse cells;    -   4. recovering lymphocyte containing cells from said mouse;    -   5. separating human lymphocytes from the recovered cells by        centrifugation; and    -   6. separating said human lymphocytes producing said antibody.    -   7. immortalizing said human lymphocytes    -   8. cloning the obtained immortalized human-derived lymphocytes        producing said antibody; and        recovering a monoclonal antibody specific to said desired        antigen from the cloned immortalized human-derived lymphocytes.

The antibodies of the present invention may be provided as part of apharmaceutical composition. Such a pharmaceutical composition may beused for treating cancer, preferably for treating leukaemia, morepreferably for treating B-CLL leukaemia, more preferably for treatingpoor prognosis B-CLL leukaemia.

Use of Antibodies in Therapy

Antibodies directed against epitopes can be used for prevention and/ortherapy of for example B-CLL. Antigenic epitopes can be used in vaccinesto stimulate an immunological response in a mammal that is directedagainst cells having the B-CLL-associated epitope found in the AMB-1protein(s) or functional equivalents. Antibodies directed against theantigenic epitopes of the invention can combat or prevent B-CLL.

An antigenic epitope may be administered to the mammal in an amountsufficient to stimulate an immunological response against the antigenicepitope. The antigenic epitope may be combined in a therapeuticcomposition and administered in several doses over a period of time thatoptimizes the immunological response of the mammal. Such anImmunological response can be detected and monitored by observingwhether antibodies directed against the epitopes of the invention arepresent in the bloodstream of the mammal.

Such antibodies can be used alone or coupled to, or combined with,therapeutically useful agents. Antibodies can be administered to mammalssuffering from any B-CLL that displays the B-CLL-associated epitope.Such administration can provide both therapeutic treatment, andprophylactic or preventative measures. For example, therapeutic methodscan be used to determine the spread of a B-CLL and lead to itsremission.

Therapeutically useful agents include, for example, leukeran, adrimycin,aminoglutethimide, aminopterin, azathioprine, bleomycin sulfate,bulsulfan, carboplatin, carminomycin, carmustine, chlorambucil,cisplatin, cyclophosphamide, cyclosporine, cytarabidine, cytosinearabinoside, cytoxin dacarbazine, dactinomycin, daunomycin,daunorubicin, doxorubicin, esperamicins, etoposide, fluorouracil,ifosfamide, Interferon-α, lomustine, melphalan, mercaptopurine,methotrexate, mitomycin C, mitotane, mitoxantrone, procarbazine HCl,taxol, taxotere (docetaxel), teniposide, thioguanine, thiotepa,vinblastine sulfate, vincristine sulfate and vinorelbine. Additionalagents include those disclosed in Chapter 52, Antineoplastic Agents(Paul Calabresi and Bruce A. Chabner), and the introduction thereto, pp.1202-1263, of Goodman and Gilman's “The Pharmacological Basis ofTherapeutics”, Eighth Edition, 1990, McGraw-Hill, Inc. (HealthProfessions Division). Toxins can be proteins such as, for example,pokeweed anti-viral protein, cholera toxin, pertussis toxin, ricin,gelonin, abrin, diphtheria exotoxin, or Pseudomonas exotoxin. Toxinmoieties can also be high energy-emitting radionuclides such ascobalt-60, I-131, I-125, Y-90 and Re-186, and enzymatically activetoxins of bacterial, fungal, plant or animal origin, or fragmentsthereof.

Chemotherapeutic agents can be used to reduce the growth or spread ofB-CLL cells and tumors that express the AMB-1 associated epitope of theinvention. Animals that can be treated by the chemotherapeutic agents ofthe invention include humans, non-human primates, cows, horses, pigs,sheep, goats, dogs, cats, rodents and the like. In all embodiments humanB-CLL antigens and human individuals are preferred.

Species-dependent antibodies can be used in therapeutic methods. Such aspecies-dependent antibody has constant regions that are substantiallynon-immunologically reactive with the chosen species. Suchspecies-dependent antibody is particularly useful for therapy because itgives rise to substantially no immunological reactions. Thespecies-dependent antibody can be of any of the various types ofantibodies as defined above, but preferably is mammalian, and morepreferably is a humanized or human antibody.

Compositions

Therapeutically useful agents can be formulated into a composition withthe antibodies of the invention and need not be directly attached to theantibodies of the invention. However, in some embodiments,therapeutically useful agents are attached to the antibodies of theinvention using methods available to one of skill in the art, forexample, standard coupling procedures.

Compositions may contain antibodies, antigenic epitopes or trypsin-likeprotease inhibitors. Such compositions are useful for detecting theAMB-1 protein (for example antigenic epitopes) and for therapeuticmethods involving prevention and treatment of B-CLLs associated with thepresence of the AMB-1 (for example antigenic epitopes).

The antibodies, (and for example antigenic epitopes and proteaseinhibitors) can be formulated as pharmaceutical compositions andadministered to a mammalian host, such as a human patient in a varietyof forms adapted to the chosen route of administration. Routes foradministration include, for example, intravenous, intra-arterial,subcutaneous, intramuscular, intraperitoneal and other routes selectedby one of skill in the art.

Solutions of the antibodies, (and for example antigenic epitopes andprotease inhibitors) can be prepared in water or saline, and optionallymixed with a nontoxic surfactant. Formulations for intravenous orintra-arterial administration may include sterile aqueous solutions thatmay also contain buffers, liposomes, diluents and other suitableadditives.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions comprising the activeingredient that are adapted for administration by encapsulation inliposomes. In all cases, the ultimate dosage form must be sterile, fluidand stable under the conditions of manufacture and storage.

Sterile injectable solutions are prepared by incorporating theantibodies, antigenic epitopes and protease inhibitors in the requiredamount in the appropriate solvent with various of the other ingredientsenumerated above, as required, followed by filter sterilization.

Polynucleotides

In a still further aspect the invention relates to an isolatedpolynucleotide selected from the group consisting of:

-   i) a polynucleotide comprising nucleotides of SEQ ID No 5,-   ii) a polynucleotide encoding a polypeptide having the amino acid    sequence of SEQ ID No 3,-   iii) a polynucleotide, the complementary strand of which hybridises,    under stringent conditions, with a polynucleotide as defined in any    of i) and ii).-   iv) a polynucleotide which is degenerate to the polynucleotide of    iii), and-   v) the complementary strand of any such polynucleotide.

The polypeptides encoded by the polynucleotides may furthermore

-   a) have at least 60% sequence identity with the amino acid sequence    of SEQ ID No 3 and have interleukin or cytokine activity,-   b) be recognised by an antibody, or a binding fragment thereof,    which is capable of recognising an epitope, wherein said epitope is    comprised within a polypeptide having the amino acid sequence of SEQ    ID No 3; and/or-   c) be competing with a polypeptide having the amino acid sequence as    shown in SEQ ID No 3 for binding to at least one predetermined    binding partner such as a cytokine receptor.

Specific examples of fragments of SEQ ID No 1 include the nucleotidesequence selected from the group consisting of SEQ ID No:12, SEQ IDNo:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17 and SEQ IDNo:18.

Hybridisation

The entire nucleotide sequence of the coding sequence of SEQ ID No 1and/or SEQ ID No 5 or portions thereof can be used as a probe capable ofspecifically hybridising to corresponding sequences. To achieve specifichybridisation under a variety of conditions, such probes includesequences that are unique and are preferably at least about 10nucleotides in length, and most preferably at least about 20 nucleotidesin length. Such probes can be used to amplify corresponding sequencesfrom a chosen organism or individual by the well-known process ofpolymerase chain reaction (PCR) or other amplification techniques. Thistechnique can be used to isolate additional nucleotide sequences from adesired organism or as a diagnostic assay to determine the presence ofthe coding sequence in an organism or individual. Examples includehybridisation screening of plated DNA libraries (either plaques orcolonies; see e.g. Innis et al. (1990) PCR Protocols, A Guide to Methodsand Applications, eds., Academic Press).

The terms “stringent conditions” or “stringent hybridisation conditions”include reference to conditions under which a probe will hybridise toits target sequence, to a detectably greater degree than other sequences(e.g., at least twofold over background). Stringent conditions aretarget sequence dependent and will differ depending on the structure ofthe polynucleotide. By controlling the stringency of the hybridisationand/or washing conditions, target sequences can be identified which are100% complementary to a probe (homologous probing).

Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing).

Generally, probes for hybridisation of this type are in a range of about1000 nucleotides in length to about 250 nucleotides in length.

An extensive guide to the hybridisation of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). See also Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

Specificity is typically the function of post-hybridisation washes, thecritical factors being the ionic strength and temperature of the finalwash solution.

Generally, stringent wash temperature conditions are selected to beabout 5° C. to about 2° C. lower than the melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The melting point,or denaturation, of DNA occurs over a narrow temperature range andrepresents the disruption of the double helix into its complementarysingle strands. The process is described by the temperature of themidpoint of transition, Tm, which is also called the meltingtemperature.

Formulas are available in the art for the determination of meltingtemperatures.

Preferred hybridisation conditions for the nucleotide sequence of theinvention include hybridisation at 42° C. in 50% (w/v) formamide, 6×SSC,0.5% (w/v) SDS, 100 mg/ml salmon sperm DNA. Exemplary low stringencywashing conditions include hybridization at 42° C. in a solution of2×SSC, 0.5% (w/v) SDS for 30 minutes and repeating. Exemplary moderatestringency conditions include a wash in 2×SSC, 0.5% (w/v) SDS at 50° C.for 30 minutes and repeating.

Exemplary high stringency conditions include a wash in 2×SSC, 0.5% (w/v)SDS, at 65° C. for 30 minutes and repeating. Sequences that correspondto the AMB-1 gene or fractions thereof according to the presentinvention may be obtained using all the above conditions. For purposesof defining the invention, the high stringency conditions are used.

Mutations

Finally, the invention provides a method for determining an increased ordecreased predisposition for B-CLL comprising determining in abiological sample from an individual a germline alteration in a targetnucleic acid sequence comprising 150,000 nucleotides, said targetnucleic acid sequence comprising at least 10 nucleotides of SEQ ID No 1and/or SEQ ID No:5. This aspect is based on the finding of theimportance of the expression product of SEQ ID No 1 and/SEQ ID No:5, andthe complete absence of any detectable expression product of SEQ ID No 1and/or SEQ ID No:5 in healthy tissue and in patients with good prognosisB-CLL. It is highly likely that the difference is caused by a germinealteration. A germline alteration can be targeted by gene therapymethods and by the methods provided in the present invention.

Preferably, said predisposition is a predisposition for poor prognosisof B-CLL.

EXAMPLES Example 1 cDNA Cloning

By Differential Display (Pardee et al., 1992, Jorgensen et al., 1999)part of a gene (hereafter referred to as AMB-1) was found that isexpressed in unmutated B-CLL patients with poor prognosis. This gene isnot found in the mutated B-CLL patients. When AMB-1 was sequenced andaligned to known sequences in GenBank, perfect homology was found to 225base pairs (bp) of human genomic DNA from chromosome 12.

RNA was prepared using the RNeasy kit from Qiagen, as described by themanufactor (Qiagen, Hilden, Germany). RNA was prepared from patientswith B-CLL without hyper mutation who, by PCR analysis, using primersFDP5 (CCTTTATGTGTGTGACAAGTG) and F10 (ATCCAGCCAGGATGAAATAGAA), showed ahigh level of the resulting PCR fragment. Poly-A⁺ RNA was isolated fromtotal RNA by the “MicroPoly(A)Purist” kit from Ambion, as described bythe manufactor (Ambion, Inc., Texas, USA). Cloning-ready cDNA wasprepared from 8 μg poly-A⁺ RNA using the “ZAP Express® XR LibraryConstruction Kit” from Stratagene as described by the manufactor(Stratagene, San Diego, USA). The cDNA was size fractionated and twosize fractions (fraction-1: >2500 bp and fraction-2 300-2500 bp) wereindependently ligated to pre-digested lambda Zap vectors and packed intophage particles as described by the manufacture (Stratagene, San Diego,USA). The titer was determined for each library and 200,000 pfu of fromthe fraction-1 library were plated onto two 22×22 cm screening plates(100,000 pfu on each plate) and 750,000 pfu of the fraction-2 librarywere plated on five 22×22 cm screening plates (150,000 pfu on each) asdescribed by Stratagene, San Diego, USA. The plates were incubated at37° C. for 18 hours and the plaques transferred to replica nylon filters(Amersham) and denatured and renatured to allow hybridisation. Allprocedures were made as described by the manufactures (Stratagene, SanDiego, USA & Amersham Biosciences, Buckinghamshire, UK).

The filters were screened by independent hybridisations withalpha[³²P]-dATP-labelled DNA fragments; alpha[³²P]-dATP was purchasedfrom Amersham Biosciences, Buckinghamshire, UK. Between succesivehybridisations, the old probe was removed by incubation for 20 min in 2l 90-100° C. water containing 0.1% SDS. The DNA fragments used as probeswere (all positions relate to sequence ID # X): 1) pos. 48978-49250; 2)pos. 50011-51591; 3) pos. 51461-52182; 4) pos. 51901-52589; 5) pos.53121-56521; 6) pos. 58163-59408. All hybridisations and washes weremade according to the instructions from Stratagene, San Diego, USA andAmersham Biosciences, Buckinghamshire, UK; Washing was done at a highstringency (0.1×SSC at 65° C. for 20 min).

A total of 38 plaques that showed a positive response from one or moreof the screenings, were excised from the screening plates and grown asplasmids as described (Stratagene, San Diego, USA).

A total of 8 cDNAs were identified by cDNA cloning or by a combinationof cDNA cloning, PCR analysis and RACE (rapid amplification of cDNAends-polymerase chain reaction) using the SMART RACE cDNA amplificationkit (Clontech, Palo Alto, Calif.) according to the manufacturer'sinstructions.

Example 2 Bioinformatic Analysis of AMB-1

The DNA and protein sequence data bases (GenBank and EBI) have beensearched for sequences with similarity to AMB1. There is no significantDNA sequence similarity to any known gene. In particular, the codingregion of the AMB1 mRNA (SEQ ID no 3) is not present in any known EST.The only significant match to the complete mRNA sequences and the DNAsequence of the putative coding region were BAC clones derived from theregion on human chromosome 12 where the gene is located. The “AMB-1gene” had not been annotated as a gene on the chromosome. Searches withthe peptide sequence in the sptrnr data base of peptide sequences(includes Sprot and nrtrembl) showed a low similarity to putative intronmaturases from cloroplasts and to bovine IL4. The percentage similarityto both maturases and bovine IL4 was low (25.6% and 30.3%, respectively)and the similarity to maturases only included a match to 75 amino acidsof the much larger maturases. In contrast, the match to bovine IL4extended over the full peptide sequence. IL4, and other 4-helicalcytokines, include a leader peptide sequence (signal peptide) allowingthe proteins to be secreted. The AMB1 peptide sequence includes aN-terminal peptide sequence with similarity to signal peptide sequences,however, it is not a typical sequence.

A 3D search has been performed, where a peptide sequence is searched forsimilarity to known protein or peptide 3D-structures. The two bestmatches were the thioredoxin fold and the human 4-helical cytokine IL4(FIG. 5). The two matches had almost similar probability scores (2.88and 3.05, respectively). Searches with 4-helical cytokine peptidesequences (IL4, IL3, IL13 and GM-CSF) revealed that all could be foldedinto both a 4-helical cytokine structure and the thioredoxin fold.Alignment based on the structural similarity between IL4 (d1iara) andAMB-1 is shown in FIG. 5. Thus, the AMB1 peptide sequence share thisproperty with 4-helical cytokines. The structural similarity is notperfect (FIG. 6) and there are no obvious glycosylation sites in theAMB1 sequence, however, the similarity is significant. Alignment of theAMB1 peptide sequence with the sequences of IL4, IL3, IL13 and GM-CSF,based on their structures, showed very little sequence conservation buta high degree of structural conservation (FIG. 7). Based on thisalignment, AMB1 has similarities to all the 4-helical cytokines, and thelength of AMB1 and the position of gaps in the alignment could suggest ahigher similarity to e.g. IL13, but searches at 3D-PSSM only identifieda significant similarity to the structure of IL4, not IL13, IL3 orGM-CSF. However, the search algorithms are not perfect and may thereforenot detect a possible low structural similarity.

Example 3 Differential Expression of AMB-1 Patient Material

Blood samples were collected from newly diagnosed untreated patientswith B-CLL. Mononuclear cells were isolated by Lymphoprep separation(Nycomed Pharma, Oslo, Norway), and the percentage of CD5+CD20+ B-CLLcells in the mononuclear fraction was >90% in all samples as determinedby flow cytometric analysis.

Isolation of RNA and Conversion to cDNA.

Material for RNA production was isolated mononuclear cells from B-CLLpatients or mononuclear cells from lymphoprep separated buffy coats fromnormal donors. Total RNA was isolated from 5×10⁷ or more cells using theQIAamp RNA Blood Mini kit (Qiagen, Valencia, Calif.) with DNAsetreatment. RNA (1 ug) was converted to cDNA by incubation with a mixtureof random-primers (1 μg) and T24-primer (1 μg) for 5 minutes at 70° C.After cooling on ice, the reaction mixture was added to a final volumeof 25 μl containing 30 U of AMV Reverse Transcriptase HC (Promega,Madison, Wis., USA), 1× First Strand Buffer (5 mM Tris-HCl, pH 8.3, 50mM KCl, 10 mM MgCl₂, 10 mM DTT, 0.5 mM spermidine), 2.5 mM of each dNTPand 60 U rRNasin ribonuclease inhibitor (Promega, Madison, Wis., USA).The reaction was performed for 60 minutes at 37° C.

Determination of Somatic Hypermutation Status

Two μl of cDNA was amplified using a GeneAmp PCR System 2700 (AppliedBiosystems, Warrington, UK) with a 40 pmol specific upstream primercorresponding to 1 of the 6 human VH family leader sequences (VH1:5′-CCATGGACTGGACCTGGAGG-3′, VH2: 5′-ATGGACATACTTTGTTCCAGC-3′, VH3:5′-CCATGGAGTTTGGGCTGAGC-3′, VH4: 5′-ATGAAACACCTGTGGTTCTT-3′, VH5:5′-ATGGGGTCAACCGCGATCCT-3′, VH6: 5′-ATGTCTGTCTCCTTCCTCAT-3′) and a 40pmol downstream primer (Cμ:5′-GAGGCTCAGCGGGAAGACCTT-3′ orCγ:5′-GGGGAAGACCGATGGGCCCCT-3′) corresponding to a consensus sequence ofthe constant region of IgM or IgG respectively. The ReverseTranscription (RT)-PCR reaction contained 1×PCR buffer (10 mM Tris-HCl,pH 9.0, 50 mM KCl, 0.1% Triton X-100), 2.5 mM MgCl₂, 0.2 mM of each dNTPand 1.5 U Taq DNA Polymerase (Promega, Madison, Wis., USA) in a finalvolume of 100 μl. The RT-PCR was performed under the followingconditions: 1 cycle of 94° C. for 5 minutes, 30 cycles of denaturationat 94° C. for 30 secs, annealing at 62° C. for 30 sec. and extension at72° C. for 30 sec, and a final extension at 72° C. for 7 minutes. TheRT-PCR products were analysed on 2% agarose gels and sequenced in an HBIPrism 310 Genetic Analyzer (Perkin Elmer, Foster City, Calif., USA)using the BigDye Terminator Cycle Sequencing Ready Reaction kit (AppliedBiosystems, Warrington, UK) following the manufacturer's Instructions.

Sequences obtained from each sample were compared to germ line sequencesin the V base sequence directory (I. M. Tomlinson, MRC Center forProtein Engineering, Cambridge, UK) using BLAST, and the closest germline sequence was assigned. A gene sequence was considered to be mutatedif it had equal or more than 2% sequence alterations when compared tothe closest published germ line sequence.

RT-PCR that Amplifies the Exon 2-Exon 3 Junction

To evaluate the mRNA expression pattern of AMB1 in unmutated and mutatedB-CLL patients RT-PCR was performed. Exon-overlapping oligonucleotideprimers were: 5′-ATCCAGCCAGGATGAAATAGAA-3′ and5′-CACTTGTCACACACATAAAGG-3′. The RT-PCR was performed in a GeneAmp PCRSystem 2700 thermal cycler with an initial denaturation at 94° C. for 2minutes, 40 cycles of 96° C. for 25 sec., 62° C. for 25 sec. and 72° C.for 90 secs, and a final extension at 72° C. for 5 minutes. Thereactions contained 2 μl cDNA, 1×DDRT-PCR buffer (10 mM Tris-HCl, pH8.3, 50 mM KCl, 1.8 mM MgCl₂, 0.1% Triton X-100, 0.005% gelatine), 0.25mM of each dNTP, 30 pmol of each primer and 0.5 U Taq DNA Polymerase(Promega, Madison, Wis., USA) in a 30 μl final volume. RT-PCR productswere analyzed by gel electrophoresis on 2% agarose gels and visualizedwith a Gene Genius Bio Imaging System (Syngene, Frederick, Md.) afterstaining with ethidium bromide. An actin control RT-PCR was performedusing the primers: 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ and5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′.

RT-PCR that Amplifies the Exon 1-Exon 3 Junction:

To evaluate the mRNA expression pattern of AMB1 in unmutated and mutatedB-CLL patients RT-PCR was performed. Exon-overlapping oligonucleotideprimers were: 5′-AGACGGCTCTCACC AATAAG-3′ and5′-CACTTGTCACACACATAAAGG-3′. The RT-PCR was performed in a GeneAmp PCRSystem 2700 thermal cycler with an initial denaturation at 94° C. for 2minutes, 40 cycles of 96° C. for 25 sec., 62° C. for 25 sec. and 72° C.for 90 secs, and a final extension at 72° C. for 5 minutes. Thereactions contained 2 μl cDNA, 1×DDRT-PCR buffer (10 mM Tris-HCl, pH8.3, 50 mM KCl, 1.8 mM MgCl₂, 0.1% Triton X-100, 0.005% gelatine), 0.25mM of each dNTP, 30 pmol of each primer and 0.5 U Taq DNA Polymerase(Promega, Madison, Wis., USA) in a 30 μl final volume. RT-PCR productswere analyzed by gel electrophoresis on 2% agarose gels and visualizedwith a Gene Genius Bio Imaging System (Syngene, Frederick, Md.) afterstaining with ethidium bromide (Continental Lab Products, San Diego,USA).

Statistical Analysis

Statistical significance of the correlation between somatichypermutation status and AMB1 expression was analyzed using Fisher'sexact test.

Northern blotting. RNA from spleen, bone marrow and colon was purchasedfrom Clontech. The AMB1 probe was an 896 base pair fragment(57661-56766) obtained by RT-PCR as described above with the primers5′-TCACCTGGGAGCTCAGAGGA-3′ and 5′-GTGATCCTGGGAGMTCTCT-3′. For Northernblotting, 5 μg of RNA was run on a 1% agarose-gel with 6% formaldehydedissolved in 1×MOPS (20 mM 3-(N-morpholino)-propane-sulfonic acid, 5 mMsodium acetate, 1 mM EDTA, pH 7.0) for size separation. The presence ofequal amounts of RNA in each lane was ensured by ethidium bromidestaining. The RNA was transferred to a Hybond-N membrane (Amersham,Little Chalfont, UK) by capillary blotting and fixed by UV-irradiation.The filters were pre-hybridized for 1-2 hours at 42° C. in 6 ml ULTRAhyb(Ambion, Austin, Tex., USA) preheated to 68° C. and hybridized overnightat 42° C. after addition of further 4 ml containing the ³²P-labeledprobe and sheared salmon sperm DNA (10 μg/ml). The membranes were washedfor 2×15 min. at 42° C. in 2×SSC, 0.1% SDS followed by 1×15 min. in0.2×SSC, 0.1% SDS and 2×15 min. in 0.1×SSC, 0.1% SDS at 42° C. The blotwas developed and quantified by a phosphoimager. The sizes of the mRNAswere determined by reference to 18S and 28S ribosomal RNA, which werevisualized by ethidium bromide staining. The AMB1 probe used forhybridization was radiolabeled with [α-³²P] dCTP using the RandomPrimers DNA Labeling System (Gibco BRL).

Dot blot of multiple tissue expression (MTE) array. An MTE array(Clontech, Palo Alto, Calif., USA) was hybridised to AMB1 at 65° C. inExpressHyb (Clontech) supplemented with sheared salmon sperm DNA (7.5μg/ml) and human C_(o)t-1 DNA (1.5 μg/ml) according to the manufacturersrecommendations. The tissue types represented on the MTE array are shownin FIG. 11. Following hybridisation the filter was washed 5×20 min. at65° C. in 2×SSC (1×SSC=150 mM NaCl, 15 mM sodium citrate, pH 7.0), 1%SDS and 2×20 min at 65° C. in 0.1×SSC, 0.5% SDS. The blot was developedand quantified by a phosphoimager (Fuji Imager Analyzer BAS-2500, ImageReader ver. 1.4E, Image Gauge ver. 3.01 software, Fuji, Stockholm,Sweden). The membranes were stripped by boiling in 0.5% SDS for 10 min.before rehybridization. The probe used for hybridization wereradiolabeled with [α-³²P] dCTP using the Random Primers DNA LabelingSystem (Gibco BRL, Rockville, ML, USA).

Results

Based on the known sequence of the AMB-1 cDNA RT-PCRs with primers thatextend across the Exon 2-Exon 3 junction and the Exon 1-Exon 3 junctionwere set up. As shown in FIG. 10, where the Exon 2-Exon 3 junction hasbeen amplified, AMB-1 is expressed in the unmutated patients (UPN1-8)while no expression of AMB-1 is seen in mutated patients (UPN9-16).

Northern blot analysis was performed to determine the size of AMB-1'smRNA transcript. As shown in FIG. 3 the probe identifies transcripts inthe samples from the three patients without somatic hypermutation (UPN1,UPN4 and UPN7). However, the probe does not recognise any transcriptsfrom the patients with somatic hypermutation (UPN9, UPN10, UPN13, UPN21)or the various cell lines and tissue samples. Similar results wereobtained when cell lines and tissue samples were investigated by RT-PCR(results not shown). Dot blot analysis on a purchased filter with 96different RNA samples (FIG. 8) only revealed specific binding to thetotal DNA control dot, but not to any specific tissue (results notshown). Thus AMB-1 is only expressed in B-CLL cells withouthypermutation or AMB-1 is expressed at extremely low levels in othertissues.

We next tested the predictive value, in terms of IgV_(H) mutationalstatus, of expression of AMB-1 in 29 consecutive newly diagnosedpatients. At present 13 somatically unmutated and 16 somatically mutatedpatients have been included in our prospective patient database. Thesensitivity and specificity for expression of AMB-1 in predictingmutational status is well above 90% (p<0.0001), which is at the levelobtained by sequencing.

Example 4 Investigation of the Prognostic Significance of AMB-1 in Termsof Patient Survival

To obtain information about the ability of AMB-1 to predict survival ortime to progression for B-CLL patients survival curves were made. Foreach patient the following data were collected: clinical stage at timeof diagnosis (Rai and Binet staging), date of diagnosis, date of firsttime of treatment and last follow up date. Time to treatment(progression free survival) and survival time were calculated based onthese dates using the Microsoft Excel software. Survival times andprogression free survival times were plotted be the Kaplan-Meier Methodand compared using the log-rank test (Prism 3.0 Graph Pad software).

34 newly diagnosed, untreated B-CLL patients were investigated asdescribed above. As shown in FIG. 2 a-d AMB-1 expression is a goodpredictor of B-CLL patient survival and B-CLL patient time toprogression.

Example 5 siRNA Assays Materials and Methods:

Frozen cells from unmutated or mutated B-CLL patients purified andcharacterized for mutational status as previously described were thawedand Ficoll separated to obtain the live cells. Cells were counted andresuspended at 2×10⁶ cells/ml in RPMI 1640 medium with glutamax-1 (RPMI,GIBCO, Paisley, UK). 1×10⁶ cells (500 μl of cell suspension) wereincubated on ice for 10 minutes with the appropriate siRNA orcombination of siRNAs at a final concentration of 100 nM each. Thesuspension was electroporated using a 4 mm cuvette (MolecularBioProducts, San Diego, Calif., USA) in a BioRad Gene Pulser. Followingelectroporation the cells were incubated on ice for 10 minutes, washedonce in RPMI with 20% FCS and 100 units/ml Penicillin and 100 μg/mlStreptomycin (GIBCO, Paisley, UK), resuspended at a concentration of4×10⁶ cells/ml in the same medium and Incubated for 48 hours. For eachpatient the optimal voltage allowing for maximum siRNA uptake wasdetermined by making a voltage curve (300-450V, 960 μF) using aFITC-labeled unspecific probe (Xeragon, Qiagen, Hilden, Germany) at afinal concentration of 200 nM and the uptake was followed by flowcytometry.

The siRNAs were produced using the Silencer™ siRNA Construction Kit,(Ambion, Texas, USA) according to the manufacturers instructions. SiRNAshad the following target sequences:

TABLE 1 Overview of the siRNAs used in electroporation experiments siRNAname: mRNA target sequence: Exon 1 A 5′-AAUAAGGGCAGGCAUCAUCCA-3′ SEQ IDNo:19 Exon 1 B 5′-AAUUACACUGCCAGGUUUCCU-3′ SEQ ID No:20 Exon 2 A5′-AAUUCAUUCACAAUGAUUGCU-3′ SEQ ID No:21 Exon 2 B5′-AAUUUCUCUUGGGUAAUUCAG-3′ SEQ ID No:22 Exon 35′-AAAAUCAGAAUCUGCGCAGCA-3′ SEQ ID No:23 (DDend) A Exon 35′-AAUGAUGAUGGGAAGAAGGAA-3′ SEQ ID No:24 (DDend) B CDS A5′-AAACUUAGUAAUUGAGUGUGA-3′ SEQ ID No:25 CDS B5′-AAUAUGUCACUUUCAUAAAGC-3′ SEQ ID No:26 Transcript5′-AAUGAUGAUGGGAAGAAGGAA-3′ on-strand A Transcript5′-AAACUAUGAGAUUUCAGAAGG-3′ on-strand B

After 48 hours of incubation the various B-CLL samples were counted andlive and dead cells were distinguished by nigrosin exclusion (0.1% inNigrosin in PBS from Fluka, Buchs, Switzerland).

Results:

Following 48 hours of incubation with control GFP siRNA or siRNAsagainst various regions of the cDNAs (see table 1), the viable cells anddead cells were counted based on the ability of the cells to excludenigrosin. As shown in table 2, in 3 out of 4 patients introduction ofsiRNAs against Exon 1, Exon 2 and Exon 3 resulted in an increase of deadcells as compared to the controls (no siRNA or GFP siRNA).

TABLE 2 Percent dead cells of total cells following electroporation andincubation with siRNAs. Treatment: UPN 67 UPN 66 UPN 62 UPN 73 No siRNA33.0% 19.7% 30.9% 20.2% Control siRNA 28.4% 26.7% 24.0% 16.0% CDS siRNA23.7% 25.5% 21.0% 13.3% Ex1-3 siRNA 47.3% 54.1% 17.2% 29.1%

Example 6 Identification of Possible Cytogenetic Aberrations Near orWithin the Region Encoding AMB-1 on Chromosome 12

Rationale: The limited expression profile of AMB-1 suggests that it maybe a result of a genetic aberration (e.g. deletion, translocation oralternative splicing) or that the promotor region controlling theexpression of AMB-1 is uniquely activated in unmutated B-CLL. Anothergene is situated about 200.000 bases upstream of the AMB-1 gene (SEQ IDNo 1) on chromosome 12 and the inventors we have determined that thisgene is expressed at equal levels in unmutated and mutated patients.

Methods: Using primers, initially spaced about 20.000 bp apart; thisregion on chromosome 12 is characterised in unmutated B-CLL patients. Ifgenetic aberrations within the region are detected by PCR analysis ofchromosomal DNA, detailed molecular genetic studies using FISH,microsatellite analysis and Southern blotting will be employed. Thewhole region from unmutated patients is sequenced.

Example 7 Polyclonal Antibodies Production of Polyclonal Antibodies:

Synthetic peptides CDLETNSEINKLIIYLFSQNNRIRF and CQVSKKHIIYSTFLSKNF weresynthesized and conjugated to KLH (K.J.Ross-Petersen Aps, Holte,Denmark). Polyclonal antibodies were produced by immunization of rabbitswith these conjugated peptides by DAKO (DAKO Cytomation A/S, Glostrup,Denmark).

Testing of Polyclonal Antibodies:

At the present time we have produced polyclonal antibodies from threerabbits that have been immunized with peptides representing predictedimmunogenic regions of the protein that can be predicted from the CDSsequence (SEQ ID No:17) (cDNA 4). The antibodies are tested in variousways. The proposed reading frame of CDS (SEQ ID No:17) is expressed in293 cells and the binding of antibodies to lanes on a western blot withnon-transfected 293 cells versus transfected 293 cells are compared. Thesize of the band in the lanes with transfected 293 cells is compared tothe size of western blot bands in lanes with proteins from B-CLLpatients. Specificity of the bands is secured by peptide blockingexperiments.

Additionally the polyclonal antibodies are tested in B-CLLimmunoprecipitation experiments where the antibodies are used toimmunoprecipitate the protein produced from the CDS sequence and theimmunoprecipitates are analyzed by western blotting.

Example 8 Assay for the Biological Activity of 4-Helix Cytokines

The assay is based on the use of a cytokine dependent or stimulated cellline, for example an IL4 dependent cell line (“Optimisation of theCT.h4S bioassay for detection of human interleukin-4 secreted bymononuclear cells stimulated by phytohaemaglutinin or by human leukocyteantigen mismatched mixed lymphocyte culture”, Petersen, S. L., Russell,C. A., Bendtzen, K. & Vindeløv, L. L., Immunology Letters 84 (2002)29-39). Other examples of cytokine dependent cell lines include IL13dependent cell lines. A list of commercially available cytokinedependent cell lines is disclosed in the general part of thedescription. These can all be used for assessing cytokine activity. Themost preferred cell lines are those that are IL4 dependent.

The assay can be performed in two ways. The first assay comprisesproviding recombinantly produced AMB1 protein or a functional equivalentthereof and determine the proliferation rate of the cell line. Theproliferation rate (either rate of proliferation or ±proliferation) canbe compared to the proliferation rate of the cell line exposed to IL4 oranother known 4-helical cytokine or interleukin.

If a positive result is obtained with a polypeptide an assay will beperformed on the same cell line with the IL4 receptor blocked. This willcheck whether the stimulus goes through IL4R.

The second assay is based on transfection of a gene encoding a 4-helixcytokine according to the invention into cytokine dependent cells andobserve proliferation or non-proliferation during transient expression.

Example 9 Cytokine Receptor Binding Assays

The following is a description of the layout of a cytokine receptorbinding assay used to determine the cytokine activity of the 4-helixcytokines according to the present invention.

The assays can be performed with any cytokine receptor. Preferredreceptors include but is not limited to the receptors for IL4 IL13, IL3,and GM-CSF.

The ability of recombinant cytokine receptor to bind to 4-helicalcytokine is assessed in a competitive binding ELISA assay as follows.Purified recombinant cytokine receptor (IL4, IL13, IL3 or GM-CSFreceptors) (20 μg/ml in PBS) is bound to a Costar EIA/RIA 96 wellmicrotiter dish (Costar Corp, Cambridge Mass., USA) in 50 μL overnightat room temperature. The wells are washed three times with 200 μL of PBSand the unbound sites blocked by the addition of 1% BSA in PBS (200μl/well) for 1 hour at room temperature. The wells are washed as above.Biotinylated AMB-1 (1 μg/ml serially diluted in twofold steps to 15.6ng/mL; 50 μL) is added to each well and incubated for 2.5 hours at roomtemperature. The wells are washed as above. The bound biotinylated AMB-1is detected by the addition of 50 μl/well of a 1:2000 dilution ofstreptavidin-HRP (Pierce Chemical Co., Rockford, Ill.) for 30 minutes atroom temperature. The wells are washed as above and 50 μL of ABTS(Zymed, Calif.) added and the developing blue color monitored at 405 nmafter 30 min. The ability of unlabelled 4-helical cytokine to competewith biotinylated AMB-1, respectively, is assessed by mixing varyingamounts of the competing protein with a quantity of biotinylated AMB-1shown to be non-saturating (i.e., 70 ng/mL; 1.5 nM) and performing thebinding assays as described above. A reduction in the signal (Abs 405nm) expected for biotinylated 4-helical cytokine indicates a competitionfor binding to immobilised cytokine receptor.

The above identified assays can be used to identify 4-helical cytokineswith similar binding affinities as AMB-1 (SEQ ID No. 3). In thecompetitive binding assays biotinylated IL4, IL13, IL3, or GM-CSF can beused to identify 4-helical cytokines which can compete with thesecytokines.

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1-41. (canceled)
 42. A nucleotide sequence selected from the groupconsisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 for use as amedicament.
 43. A transcriptional product selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 for use as amedicament.
 44. An amino acid sequence selected from the groupconsisting of: i) an amino acid sequence of SEQ ID NO: 3, ii) an aminoacid sequence having at least 60% sequence identity compared to the fulllength sequence of SEQ ID NO: 3 iii) a fragment of SEQ ID NO: 3 havingat least 60% sequence identity compared to the full length sequence ofSEQ ID NO: 3 for use in a diagnostic method for a subtype of B-CLLhaving a poor prognosis.
 45. An amino acid sequence according to claim44, said amino acid sequence being a polypeptide having interleukin orcytokine activity.
 46. The amino acid sequence according to any one ofclaims 44 or 45, which folds as a 4-helical cytokine.
 47. Use of anamino acid sequence as defined in any one of claims 44-46 for thepreparation of a medicament for the treatment of cancer.
 48. Useaccording to claim 47, wherein the cancer is B-CLL.
 49. A method ofimmunisation of a patient in need thereof against B-CLL, wherein saidimmunisation generates an immune response in said patient whichrecognises a translational product of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQID NO:
 11. 50. A method for producing an antibody with specificityagainst an isolated polypeptide selected from i) an amino acid sequenceof SEQ ID NO: 3, ii) an amino acid sequence having at least 60% sequenceidentity with the full length SEQ ID NO: 3, iii) a fragment of SEQ IDNO: 3 having at least 60% sequence identity compared to full length SEQID NO:
 3. 51. The method of claim 50 wherein said isolated polypeptidehas interleukin or cytokine activity.
 52. The method of claim 50 whereinthe isolated polypeptide folds as a 4-helical cytokine.
 53. An antibodyobtainable by the method of claim
 50. 54. An antibody with specificityagainst an isolated polypeptide comprising an amino acid sequenceselected from the group consisting of: i) an amino acid sequence of SEQID NO: 3, ii) an amino acid sequence having at least 60% sequenceidentity compared to the full length sequence of SEQ ID NO:3 iii) afragment of SEQ ID NO:3 having at least 60% sequence identity comparedto the full length sequence of SEQ ID NO:3.